PROJECT SUMMARY All viruses require a number of host factors to infect and replicate in human cells. RNA modifying enzymes have recently emerged as a class of factors that are likely to be broadly important in viral infection. This Exploratory and Developmental Research Proposal will explore the role of RNA modification by pseudouridylation in representative pathogenic members of the Flaviviridae. This work is motivated by the discovery of a functional requirement for the pseudouridine synthase PUS7L during the hepatitis C virus (HCV) life cycle (Marceau et al. Nature 2016), an unexplained requirement that we have confirmed in our laboratory. PUS proteins catalyze the isomerization of uridine to pseudouridine, an abundant modified nucleotide that can affect RNA structure, RNA-protein interactions, and cellular recognition of foreign RNA by innate immune sensors ? all of which have the potential to significantly impact viral infection. Here we propose to define the host and viral pseudouridine landscapes of human hepatocytes infected with HCV, DENV and ZIKV by using Pseudo-seq, a method developed in our laboratory for transcriptome-wide detection of ? with single-nucleotide resolution. With this method, we discovered that multiple PUS proteins target mRNAs for regulated pseudouridylation in addition to constitutively modifying their canonical non-coding RNA targets (Carlile et al. Nature 2014). We have recently obtained preliminary evidence that the HCV RNA genome is pseudouridylated at multiple positions, the first evidence for pseudouridine in a human pathogenic virus. We will extend these studies to DENV and ZIKV to determine whether pseudouridylation of viral genomes is common among Flaviviridae and to identify host pseudouridylation events that are common responses to viral challenge versus any that are specific to one infectious agent. We will leverage our recent success in recapitulating site-specific pseudouridylation by human PUS proteins in a high-throughput format in vitro to efficiently identify which of the 13 PUS enzymes is responsible for each viral and host RNA pseudouridine. Finally, we will investigate the functional importance of viral and host RNA pseudouridylation using PUS knockout/knockdown cell lines and rescue constructs. Completion of our aims will provide the first view of RNA pseudouridylation in the context of infection by human pathogenic viruses and may identify cellular PUS proteins as new therapeutic targets for combatting flavivirus infections.